Phospho-CREB1 (Thr100) Antibody

What is Phospho-CREB1 (Thr100) and what is its role in cellular signaling pathways?

Phospho-CREB1 (Thr100) refers to the Cyclic AMP-responsive element-binding protein 1 when phosphorylated at the threonine residue at position 100. CREB1 functions as a phosphorylation-dependent transcription factor that stimulates transcription upon binding to the DNA cAMP response element (CRE), a sequence present in many viral and cellular promoters.

While Ser133 phosphorylation is the most well-studied modification of CREB1, phosphorylation at different sites including Thr100 can distinctly regulate CREB1 activity. The protein is involved in multiple cellular processes including:

• Synchronization of circadian rhythmicity

• Differentiation of adipose cells

• Neuronal plasticity and cognitive functions

• Cell proliferation and survival

• Glucose homeostasis

• Regulation of apoptotic and inflammatory response factors in cardiomyocytes

Research indicates that phosphorylation at different KID (kinase-inducible domain) serine residues can have opposing effects: phosphorylation of Ser129 and Ser133 positively affects transcriptional activity, while phosphorylation at Ser111 and Ser121 inhibits CREB-related gene expression .

How does Phospho-CREB1 (Thr100) antibody specificity differ from antibodies targeting other phosphorylation sites?

Phospho-CREB1 (Thr100) antibodies are specifically designed to detect CREB1 protein only when phosphorylated at threonine 100. This high specificity allows researchers to distinguish this particular phosphorylation state from other modified forms of CREB1. Key differences include:

• Target specificity: Both monoclonal and polyclonal antibodies against Phospho-CREB1 (Thr100) detect "endogenous levels of CREB-1 protein only when phosphorylated at T100"

• Epitope region: The immunogen typically spans amino acids 71-120 of human CREB1, surrounding the Thr100 phosphorylation site

• Functional significance: Unlike the well-characterized Ser133 phosphorylation (which is critical for activation), the specific downstream effects of Thr100 phosphorylation are still being elucidated

When selecting between different phospho-specific antibodies, researchers should consider the particular signaling pathway under investigation and the specific phosphorylation events known to occur in response to their experimental conditions.

What are the validated applications for Phospho-CREB1 (Thr100) antibodies?

Based on manufacturer specifications and research literature, Phospho-CREB1 (Thr100) antibodies have been validated for the following applications:

When using these antibodies for new applications or sample types, researchers should perform their own validation experiments. The observed band for CREB1 is typically around 42-43 kDa .

What is the recommended protocol for detecting Phospho-CREB1 (Thr100) in Western blotting?

For optimal detection of Phospho-CREB1 (Thr100) by Western blotting, follow these methodological guidelines:

• Sample preparation:

  • Extract proteins using phosphatase inhibitor-containing lysis buffers

  • Maintain samples at 4°C throughout processing

  • Avoid repeated freeze-thaw cycles which may degrade phosphoproteins

• Gel electrophoresis and transfer:

  • Load 20-50 μg of total protein per lane (or optimize based on your samples)

  • Use freshly prepared transfer buffers

  • Consider wet transfer for optimal results with phosphoproteins

• Blocking and antibody incubation:

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Incubate with Phospho-CREB1 (Thr100) antibody at 1:500-1:2000 dilution overnight at 4°C

  • If planning to strip and reprobe, always probe for the phosphoprotein first

• Controls and validation:

  • Include positive controls (stimulated cells known to induce Thr100 phosphorylation)

  • Consider phosphatase treatment as a negative control

  • If signal is weak, concentrate the antigen by immunoprecipitation

• Detection considerations:

  • For low abundance phosphoproteins, use high-sensitivity ECL substrates

  • Consider fluorescent multiplexing to detect total and phosphorylated CREB1 simultaneously

Phosphoproteins are often present at low stoichiometric abundance, requiring careful optimization of detection methods .

How can I validate the specificity of Phospho-CREB1 (Thr100) antibody in my experimental system?

Validating antibody specificity is crucial for reliable results when working with phospho-specific antibodies. Consider these approaches:

• Phosphatase treatment:

  • Treat one set of samples with lambda phosphatase

  • Compare to untreated samples - signal should disappear in treated samples

• Peptide competition assay:

  • Pre-incubate antibody with phosphorylated versus non-phosphorylated peptides

  • Signal should be blocked by phosphorylated but not by non-phosphorylated peptide

• Genetic validation:

  • Use CREB1 knockout/knockdown models

  • Employ site-directed mutagenesis to create T100A mutants

• Physiological validation:

  • Use activators and inhibitors of pathways known to affect CREB1 phosphorylation

  • Compare results across multiple antibody sources if possible

• Cross-reactivity assessment:

  • Test the antibody against closely related proteins (e.g., ATF1)

  • Analyze samples from multiple species if working across models

Manufacturers typically specify that these antibodies detect "endogenous levels of CREB-1 protein only when phosphorylated at T100" , but independent validation in your specific experimental system is always recommended.

What are the critical considerations for sample preparation to preserve Phospho-CREB1 (Thr100)?

Phosphorylation is a labile post-translational modification that requires careful sample handling to preserve. Follow these guidelines:

• Cell/tissue harvesting:

  • Minimize the time between stimulus and harvest

  • Rapidly freeze samples in liquid nitrogen if immediate processing isn't possible

• Lysis buffer components:

  • Include multiple phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Add protease inhibitors to prevent protein degradation

  • Consider commercial phosphatase inhibitor cocktails for comprehensive coverage

• Processing conditions:

  • Maintain samples at 4°C throughout extraction

  • Avoid alkaline pH which can promote dephosphorylation

  • Process samples quickly to minimize exposure to endogenous phosphatases

• Storage considerations:

  • Store lysates at -80°C

  • Avoid repeated freeze-thaw cycles

  • Consider adding fresh phosphatase inhibitors upon thawing

• Handling antibodies:

  • Store antibodies according to manufacturer recommendations (-20°C)

  • Avoid repeated freeze-thaw cycles of antibody solutions

  • Prepare working dilutions fresh when possible

Remember that different phosphorylation sites may have different stabilities; some phosphorylation events are more labile than others.

How does phosphorylation at Thr100 affect CREB1's binding to DNA compared to other phosphorylation sites?

The effect of Thr100 phosphorylation on CREB1's DNA-binding capacity differs from that of other phosphorylation sites:

• Binding affinity differences:

  • Research has identified two classes of CRE (cAMP response element) sites with different affinities for CREB

  • Low-affinity sites (asymmetric CGTCA sites) and high-affinity sites (symmetrical TGACGTCA sites)

  • Phosphorylation increases binding to both types of sites, but the effect is more pronounced for low-affinity sites

• Comparison with Ser133 phosphorylation:

  • Ser133 phosphorylation is well-established as critical for transcriptional activation

  • Ser133 phosphorylation does not directly affect DNA binding but recruits the coactivator CREB binding protein (CBP)

  • In contrast, the specific effect of Thr100 phosphorylation on coactivator recruitment is less well-characterized

• Functional consequences:

  • Different phosphorylation patterns may affect the spectrum of genes regulated

  • Phosphorylation-dependent changes in binding can increase occupancy of weak binding sites used for transactivation

  • High-affinity sites may have constitutive binding of both transcriptionally active and inactive CREB dimers

The complex interplay between multiple phosphorylation sites likely allows for fine-tuned control of gene activation in response to different cellular stimuli.

What is the emerging role of Phospho-CREB1 (Thr100) in cancer research and potential therapeutic applications?

Research on Phospho-CREB1 has revealed significant implications for cancer biology and therapeutic strategies:

These findings suggest that phosphorylated CREB1 detection may serve both as a biomarker and potential therapeutic target in certain cancers.

How can researchers optimize detection of low-abundance Phospho-CREB1 (Thr100) in clinical samples?

Detecting low-abundance phosphoproteins in clinical samples presents significant challenges that require specialized approaches:

• Sample enrichment strategies:

  • Immunoprecipitation before Western blotting to concentrate the antigen

  • Phosphoprotein enrichment using metal oxide affinity chromatography (MOAC)

  • Subcellular fractionation to isolate nuclear proteins where CREB1 primarily functions

• Signal amplification methods:

  • Use highly sensitive detection systems such as SuperSignal West Femto

  • Consider tyramide signal amplification for immunohistochemistry

  • Explore digital immunoassay platforms with single-molecule detection capabilities

• Quantitative approaches:

  • Phospho-protein arrays for multiplex analysis

  • HTRF (Homogeneous Time Resolved Fluorescence) assays for quantitative detection

  • Phospho-flow cytometry for single-cell analysis when applicable

• Data analysis considerations:

  • Use image analysis software to quantify subtle differences in signal intensity

  • Apply appropriate statistical methods for comparing low-abundance targets

  • Consider normalization to total protein rather than housekeeping proteins

• Technical controls:

  • Include gradient standards of known phosphoprotein concentrations

  • Process all clinical samples identically and simultaneously when possible

  • Use internal controls within each sample batch

For clinical samples specifically, the pre-analytical variables (collection, storage, processing time) should be strictly controlled and documented to ensure reproducibility.

What are the key methodological differences between using polyclonal versus monoclonal Phospho-CREB1 (Thr100) antibodies?

Researchers should consider these key differences when selecting between polyclonal and monoclonal Phospho-CREB1 (Thr100) antibodies:

For studies requiring absolute specificity, monoclonal antibodies may be preferred. For maximum sensitivity or detection of multiple isoforms, polyclonal antibodies might offer advantages. Both antibody types are purified by affinity chromatography using epitope-specific immunogens .

What are common technical issues when using Phospho-CREB1 (Thr100) antibodies and how can they be resolved?

Researchers using Phospho-CREB1 (Thr100) antibodies commonly encounter these technical challenges:

• Weak or absent signal:

  • Causes: Low phosphorylation levels, rapid dephosphorylation during processing, insufficient antibody concentration

  • Solutions: Enrich samples via immunoprecipitation , strengthen phosphatase inhibition, increase antibody concentration or incubation time

• High background:

  • Causes: Insufficient blocking, cross-reactivity, high antibody concentration

  • Solutions: Optimize blocking conditions, use BSA instead of milk for blocking , titrate antibody concentration, increase washing stringency

• Multiple bands/non-specific binding:

  • Causes: Cross-reactivity with related proteins (e.g., ATF1), degradation products, non-specific binding

  • Solutions: Use knockout/knockdown controls, include peptide competition controls, optimize antibody dilution

• Inconsistent results between experiments:

  • Causes: Variable phosphorylation status, inconsistent sample handling, antibody degradation

  • Solutions: Standardize stimulation protocols, establish consistent sample processing workflow, aliquot antibodies to avoid freeze-thaw cycles

• Poor reproducibility in clinical samples:

  • Causes: Pre-analytical variables, tissue heterogeneity, phosphatase activity in archived samples

  • Solutions: Standardize collection and fixation protocols, use phosphatase inhibitors throughout processing, consider laser microdissection for heterogeneous tissues

For specific methodological approaches, always refer to the manufacturer's recommendations regarding optimal dilutions and application-specific protocols .

How should appropriate controls be designed for experiments using Phospho-CREB1 (Thr100) antibodies?

Robust control strategies are essential for reliable interpretation of results when working with phospho-specific antibodies:

• Positive controls:

  • Cells/tissues treated with agents known to induce CREB1 Thr100 phosphorylation

  • Recombinant phosphorylated CREB1 protein (if available)

  • Previously validated samples with confirmed Thr100 phosphorylation

• Negative controls:

  • Phosphatase-treated samples to remove phosphorylation

  • Cells treated with inhibitors of pathways leading to CREB1 phosphorylation

  • CREB1 knockout/knockdown samples (accounting for potential antibody cross-reactivity)

• Specificity controls:

  • Peptide competition with phosphorylated and non-phosphorylated peptides

  • Comparison of results using multiple antibodies targeting the same phospho-site

  • Western blot with both phospho-specific and total CREB1 antibodies

• Loading and transfer controls:

  • When analyzing phosphorylation, always probe for total CREB1 after phospho-CREB1 (after stripping)

  • Include loading controls like total protein staining (preferred) or housekeeping proteins

  • Consider multiplexing with fluorescent detection to analyze phospho-CREB1 and total CREB1 simultaneously

• Method-specific controls:

  • For Western blotting: molecular weight markers

  • For IHC: isotype control antibodies

  • For phospho-protein arrays: both positive and negative reference spots

When performing quantitative analyses, use the ratio of phosphorylated to total protein rather than absolute phospho-protein levels alone.

What are the considerations for storing and handling Phospho-CREB1 (Thr100) antibodies to maintain optimal performance?

Proper storage and handling of phospho-specific antibodies is critical for maintaining their performance over time:

• Storage temperature:

  • Store at -20°C for up to 1 year from receipt date

  • Avoid storing in frost-free freezers (temperature fluctuations)

  • Consider dedicated antibody storage freezers

• Formulation:

  • Typically supplied in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

  • This formulation helps maintain antibody stability during freeze-thaw cycles

  • Do not dilute stock solution unless preparing working dilution

• Aliquoting practices:

  • Upon receipt, divide into small single-use aliquots to avoid repeated freeze-thaw cycles

  • Use sterile tubes for aliquoting

  • Label clearly with antibody details, concentration, and date

• Working dilution handling:

  • Prepare fresh working dilutions whenever possible

  • If storing diluted antibody, keep at 4°C for short periods (≤1 week)

  • Add preservatives like sodium azide (0.02%) to diluted solutions

• Shipping and temporary storage:

  • If temporarily stored at 4°C upon receipt, transfer to -20°C as soon as possible

  • Monitor for any signs of precipitation or contamination

  • Document receipt date to track antibody age

• Contamination prevention:

  • Use clean pipette tips when accessing antibody stock

  • Avoid introducing bacteria or other contaminants

  • Consider adding additional preservatives if contamination is a concern

Following these guidelines will help maintain antibody performance and extend its useful life in the laboratory.

How can researchers design experiments to study the kinetics of CREB1 phosphorylation at Thr100 in response to specific stimuli?

Designing kinetic studies of phosphorylation events requires careful experimental planning:

• Temporal considerations:

  • Establish appropriate time points (typically ranging from seconds to hours)

  • Include both early (30 seconds, 2, 5, 15 minutes) and late (30 minutes, 1, 2, 6, 24 hours) time points

  • Use synchronized cell populations when possible

• Stimulation protocols:

  • Identify appropriate stimuli known to affect CREB1 phosphorylation

  • Optimize stimulus concentration through dose-response experiments

  • Ensure consistent application of stimulus across experimental replicates

  • Consider physiologically relevant stimuli for your model system

• Inhibitor studies:

  • Use pathway-specific inhibitors to dissect kinase contributions

  • Apply inhibitors at defined time points to study persistence of phosphorylation

  • Include appropriate vehicle controls

• Sample collection strategy:

  • Develop rapid harvesting protocols to capture transient phosphorylation events

  • Use lysis buffers optimized for phosphoprotein preservation

  • Process all time points identically to avoid technical artifacts

• Quantification approaches:

  • Normalize phospho-CREB1 signal to total CREB1

  • Consider multi-parameter analysis to correlate with upstream and downstream events

  • Use appropriate software for densitometric analysis with statistical validation

• Visual representation:

  • Plot phosphorylation levels against time

  • Include error bars representing biological replicates

  • Consider mathematical modeling to describe phosphorylation/dephosphorylation kinetics

This experimental design allows for comprehensive characterization of the temporal dynamics of Thr100 phosphorylation and its relationship to cellular responses.

How can researchers integrate Phospho-CREB1 (Thr100) analysis with other phosphorylation sites to understand complex signaling networks?

Integrative approaches provide deeper insights into signaling networks and contextual understanding of specific phosphorylation events:

• Multi-phosphorylation site analysis:

  • Compare phosphorylation patterns at multiple sites (e.g., Thr100, Ser133, Ser142, Ser111, Ser121)

  • Determine whether phosphorylation events occur sequentially or independently

  • Identify potential priming phosphorylation events that influence subsequent modifications

• Pathway integration strategies:

  • Map upstream kinases responsible for specific phosphorylation events

  • Identify cross-talk between pathways affecting different phosphorylation sites

  • Study phosphatase activity to understand negative regulation

• Correlation with functional outcomes:

  • Link specific phosphorylation patterns to functional responses

  • Assess gene expression changes correlated with different phosphorylation states

  • Study protein-protein interactions dependent on phosphorylation status

• Technological integration:

  • Combine antibody-based detection with mass spectrometry

  • Integrate cellular imaging with biochemical assays

  • Utilize computational modeling to predict network behavior

• Multi-omics approaches:

  • Correlate phosphoproteomic data with transcriptomic changes

  • Integrate with chromatin immunoprecipitation to assess DNA binding

  • Connect metabolomic changes with signaling events

Research has shown that different phosphorylation sites on CREB can have opposing effects on its activity. For example, while phosphorylation of Ser129 and Ser133 positively affects CREB transcription activity, phosphorylation at Ser111 and Ser121 can block CREB-related gene expression . Understanding these complex interactions requires integrative approaches.

What are the emerging applications of Phospho-CREB1 (Thr100) antibodies in neuroscience and developmental biology research?

Phospho-CREB1 antibodies are finding expanding applications in specialized research fields:

• Neuroscience applications:

  • Studying neuronal plasticity mechanisms

  • Investigating learning and memory processes at the molecular level

  • Examining circadian rhythm regulation where CREB plays a critical role

  • Research on neurodegenerative conditions like Alzheimer's Disease

  • Analyzing neuronal responses to stimuli in specific brain regions

• Developmental biology:

  • Tracking signaling changes during cellular differentiation

  • Studying adipose tissue development where CREB is implicated

  • Examining embryonic development of specific tissues

  • Investigating stem cell differentiation pathways

• Disease-specific applications:

  • Cancer progression and metastasis studies

  • Metabolic disorder research

  • Inflammatory response investigations

  • Cardiovascular disease models where CREB regulates apoptotic and inflammatory factors

• Specialized research approaches:

  • Spatial analysis of phosphorylation in tissue sections

  • Temporal dynamics during developmental processes

  • Single-cell analysis of phosphorylation heterogeneity

Studies have shown CREB phosphorylation in specific regions of the rat spinal cord in response to stimuli, with significant changes observed in different spinal laminae . This type of region-specific phosphorylation analysis represents an important application in specialized fields.

How can bioinformatic approaches complement experimental studies using Phospho-CREB1 (Thr100) antibodies?

Bioinformatic approaches can significantly enhance experimental phospho-CREB1 research:

• Phosphorylation site prediction:

  • Identify potential novel phosphorylation sites

  • Predict kinases responsible for specific phosphorylation events

  • Compare conservation of phosphorylation sites across species

• Network analysis:

  • Map protein-protein interaction networks affected by phosphorylation

  • Identify signaling hubs connected to CREB1 phosphorylation

  • Perform pathway enrichment analysis to understand broader context

• Transcriptional target prediction:

  • Identify genes containing CREB binding sites

  • Predict differential regulation based on phosphorylation status

  • Integrate with public ChIP-seq datasets

• Structural biology integration:

  • Model the structural impact of Thr100 phosphorylation

  • Predict allosteric effects on protein function

  • Simulate molecular dynamics with different phosphorylation states

• Clinical data integration:

  • Correlate phosphorylation patterns with patient outcomes

  • Identify potential biomarkers based on phosphorylation signatures

  • Perform survival analysis with phosphorylation data

• High-throughput data analysis:

  • Process phosphoproteomic mass spectrometry data

  • Analyze microscopy images for spatial phosphorylation patterns

  • Develop custom algorithms for kinetic modeling